Matrilin-2 within a three-dimensional lysine-modified chitosan porous scaffold enhances Schwann cell migration and axonal outgrowth for peripheral nerve regeneration

Abstract

Background: Matrilin-2 is a key extracellular matrix protein involved in peripheral nerve regeneration. We sought to develop a biomimetic scaffold to enhance peripheral nerve regeneration by incorporating matrilin-2 within a chitosan-derived porous scaffold. We hypothesized that the use of such a novel biomaterial delivers microenvironmental cues to facilitate Schwann cell (SC) migration and enhance axonal outgrowth during peripheral nerve regeneration. Materials and Methods: The effect of matrilin-2 on SC migration was evaluated with agarose drop migration assay on matrilin-2 coated dishes. SC adhesion was determined with SCs cultured atop tissue culture dishes coated with matrilin-2. Various formulations of chitosan vs matrilin-2 in scaffold constructs were examined with scanning electron microscopy. The effect of the matrilin-2/chitosan scaffold on SC migration in the collagen conduits was determined by capillary migration assays. Neuronal adhesion and axonal outgrowth were evaluated with three-dimensional (3D) organotypic assay of dorsal root ganglions (DRG). DRG axonal outgrowth within the scaffolds was determined by immunofluorescence staining of neurofilaments. Results: Matrilin-2 induced SC migration and enhanced its adhesion. A formulation of 2% chitosan with matrilin-2 demonstrated an optimal 3D porous architecture for SC interaction. Matrilin-2/chitosan scaffold enabled SCs to migrate against gravity within conduits. Chemical modification of chitosan with lysine (K-chitosan) further improved DRG adhesion and axonal outgrowth than the matrilin-2/chitosan scaffold without lysine modification. Conclusion: We developed a matrilin-2/K-chitosan scaffold to mimic extracellular matrix cues and provide a porous matrix to enhance peripheral nerve regeneration. Taking advantage of matrilin-2's capability to stimulate SC migration and adhesion, we formulated a porous matrilin-2/chitosan scaffold to support axongal outgrowth. Chemical modification of chitosan with lysine further improved matrilin-2 bioactivity in the 3D scaffold. The 3D porous matrilin-2/K-chitosan scaffolds have high potential for enhancing nerve repair by stimulating SC migration, neuronal adhesion, and axonal outgrowth.

Keywords: axon; chitosan; lysine; matrilin-2; nerve regeneration; porous; scaffold; schwann cell.

FIGURE 1

(A) Initial array of materials with tensile strength and Young’s Modulus as constraints. Materials highlighted include Polyglycolic Acid (PGA), Polycaprolactone (PCL), collagen, and chitosan. (B) Chitosan structure (Left) and chemical modification of chitosan by Lysine (K-chitosan).

FIGURE 2

A-C, S16 SC agarose drop migration assay visualized by phase-contrast microscopy. Tissue glass coverslips coated with (A) ribonuclease-free water or (B) matrilin-2. Scale bar: 20 μm. (C) Quantification of migration distances noting significantly increased migration atop matrilin-2 compared to water. * denotes statistical significance of p < 0.0001. D-F, Fluorescent microscopy of Schwann cell adherence on tissue glass covered with (D) ribonuclease-free water and (E) matrilin-2. (F) Fluorescent profile denoting cellular presence was measured with Matrilin-2 compared against water noting a significantly greater presence of fluorescence and cellular adherence with matrilin-2. * denotes statistical significance of p < 0.01.

FIGURE 3

Scanning electron microscopy of (A) empty collagen conduit, (B) collagen conduit filled with matrilin-2 and 2% chitosan, and (C) collagen conduit filled with matrilin-2 and 3% chitosan. (D) Magnification of the matrilin-2 and 2% chitosan scaffold detailing porous structures measuring around 50 μm.

FIGURE 4

(A) Schematic of a modified capillary migration study that demonstrated negligible upward migration of Schwann cells in (B) empty collagen conduit (CC) while (C) Matrilin-2/Chitosan scaffold (M2/Ch) demonstrated migration of fluorescently labeled Schwann cells. (D) Quantification of distances traveled noted an average distance of 383.43 μm.

FIGURE 5

Scanning electron microscopy with overview of collagen conduit and scaffold components: (A) 2% chitosan, (C) matrilin-2/chitosan, (E) 2% chitosan modified with lysine (K-chitosan), (G) matrilin-2/K-chitosan. Scanning electron microscopy with focus on porosity of varying scaffold structures and components: (B) 2% chitosan with a dense and gapped matrix, (D) matrilin-2/chitosan with a more porous matrix, (F) K-chitosan with a dense and less gapped matrix, and (H) matrilin-2/K-chitosan a more 3D porous network.

FIGURE 6

H&E staining of conduit demonstrating (A) collagen conduit with chitosan scaffold, (B) collagen conduit matrilin-2/chitosan scaffold, (C) collagen conduit with matrilin-2/K-chitosan scaffold with dorsal root ganglion (DRG) [black box]. (D) DRG at higher power adhere to matrilin-2/K-chitosan scaffold within collagen conduit.

FIGURE 7

Immunofluorescent imaging of matrilin-2/chitosan and matrilin-2/K-chitosan scaffold. (A) DAPI staining of neuronal cells adhered to end of conduit with matrilin-2/chitosan scaffold and (B) neurofilament staining of matrilin-2/chitosan demonstrating minimal axonal outgrowth from neuronal cells seen of same DAPI field. (C) DAPI staining of cluster of neuronal cells adhered to the end of conduit and matrilin-2/K-chitosan scaffold and (D) neurofilament staining of same DAPI field demonstrating axonal outgrowth. Scale marked at 100uM.